Therapeutic morpholino-substituted compounds

ABSTRACT

Morpholino-substituted pyridopyrimidine, quinolone, and benzopyranone derivatives inhibit phosphoinositide (PI) 3-kinase, an enzyme that regulates platelet-adhesion processes. As a consequence, the compounds in question have anti-thrombotic activity, as well as other pharmaceutical properties. The compounds claimed are represented by formula (I), (II) and (III). PI 3-kinase generates 3-phosphorylated PI second messengers which stimulate platelet adhesion under blood-flow conditions. Because platelet adhesion is a necessary step in the formation of a thrombus, inhibition by these compounds of PI 3-kinase under such conditions inhibits or prevents thrombus formation. The compounds are useful in treating PI 3-kinase-dependent conditions including cardiovascular diseases such as coronary artery occlusion, stroke, acute coronary syndrome, acute myocardial infarction, vascular restenosis, atherosclerosis, and unstable angina; respiratory diseases such as asthma, chronic obstructive pulmonary diseases (COPD), and bronchitis; inflammatory disorders; neoplasms including cancers such as glioma, prostate cancer, small cell lung cancer, and breast cancer; and diseases linked to disordered white blood cell function, such as autoimmune and inflammatory diseases.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is broadly concerned with anti-thromboticmorpholino-substituted compounds and corresponding methods of use. Moreparticularly, the present invention relates to morpholino-substitutedpyridopyrimidine, quinolone, and benzopyranone derivatives which inhibitthe enzyme phosphoinositide (PI) 3-kinase, and which are useful intreating PI 3-kinase-dependent conditions, including cardiovasculardiseases, respiratory diseases, inflammatory disorders, neoplasms suchas cancers, and diseases linked to disordered white blood cell function.

2. Description of the Related Art

Cell-adhesion interactions are crucial for a broad range ofphysiological processes, including inflammation, immunity, andhemostasis. Platelets are specialized adhesive cells which play afundamental role in the hemostatic process. Upon vascular injury,platelets adhere to specific subendothelial adhesive proteins, such asvon Willebrand factor (vWF). The binding of vWF to its specific receptoron the platelet surface, glycoprotein (GP) Ib/V/IX, induces plateletactivation and cytoskeletal reorganization. These cytoskeletal changesresult in filopodial extension and the formation of lamellipodialsheets, which are essential processes for platelet spreading and theformation of the primary hemostatic platelet plug.

An exaggerated platelet adhesion response at sites of atheroscleroticplaque rupture commonly leads to the formation of vaso-occlusiveplatelet thrombi. The formation of these thrombi in the coronary orcerebral circulation leads to heart attacks and strokes, respectively,which combined represent the lending causes of death in theindustrialized world. Platelet thrombus formation, also leads to anumber of other clinical states including unstable angina, sudden death,transient ischemic attacks, amaurosis fugax, and acute ischemia of limbsand internal organs.

Undesirable thrombosis also may be associated, however, with invasivemedical procedures such as cardiac surgery (e.g., angioplasty),abdominothoracic surgery, arterial surgery, deployment of animplementation (e.g., a stent or catheter), and endarterectomy.Furthermore, thrombosis may accompany various thromboembolic disordersand coagulopathies such as a stroke, pulmonary embolism (e.g., atrialfibrillation with embolization) and disseminated intravascularcoagulation. An unwanted thrombus also can arise from manipulation ofbody fluids, as occurs in the context of blood transfusion or fluidsampling, as well as in procedures involving extracorporeal circulation(e.g., cardiopulmonary bypass surgery) and dialysis.

Anti-coagulants and anti-platelet agents are frequently used toalleviate thrombosis. Blood clotting can be minimized or eliminated inmany instances by administering a suitable anti-coagulant, including oneor more of a coumarin derivative (e.g., warfarin and dicumarol) or acharged polymer (e.g., heparin, hirudin or hirulog), or through the useof an anti-platelet agent (e.g., aspirin, clopidogrel, ticlopidine,dipyridimole, or one of several GPIIb/IIIa receptor antagonists). Butanti-coagulants and platelet inhibitors can have side effects such ashemorrhaging, re-occlusion, “white-clot” syndrome, irritation, birthdefects, thrombocytopenia, and hepatic dysfunction. Moreover, long-termadministration of anti-coagulants and platelet inhibitors canparticularly increase risk of life-threatening illness or hemorrhage.

SUMMARY OF THE INVENTION

To avoid the aforementioned drawbacks in using anti-coagulants oranti-platelet drugs to inhibit or prevent undesirable thrombosis, it isan object of the present invention to provide an anti-thromboticmorpholino-substituted pyridopyrimidine derivative having the followingformula:

R is H, OH, F, Cl, Br, I, C₁-C₆ alkyl, aryl or (CH₂)_(n)-aryl;

-   R¹ is H, OH, F, Cl, Br, I, C₁-C₆ alkyl, C₃-C₆ cycloalkyl,    CH═CH-aryl, C≡C aryl, (CHR³)_(n)-aryl, NR³—C₁-C₆ alkyl,    NR³-cycloalkyl, NR³—(CHR³)_(n)-aryl, (CHR³)_(n)—NR³-aryl,    (CHR³)_(n)—NR³-alkyl, (CHR³)_(n)—NR³-cycloalkyl, (CHR³)C—O-aryl,    (CHR³)_(n)—O-alkyl, (CHR³)_(n)—O-cycloalkyl, O—(CHR³)_(n)-aryl,    S—(CHR³)_(n)-aryl, or CO-aryl, wherein n is 0, 1, or 2 and alkyl,    cycloalkyl or aryl is optionally substituted with F, Cl, Br, I, CN,    CO₂H, CO₂R³, NO₂, CF₃, substituted or unsubstituted C₁-C₆ alkyl,    substituted or unsubstituted cycloalkyl, substituted or    unsubstituted aryl, OCF₃, OR³, OSO₂-aryl, substituted or    unsubstituted amine, NHCOR³, NHSO₂R³, CONHR³, or SO₂NHR³; and-   R³ is H, or substituted or unsubstituted C₁-C₆alkyl, substituted or    unsubstituted aryl.

Preferred groups represented by R¹ include —CH₃, Br,

It is another object of the present invention to provide ananti-thrombotic morpholino-substituted quinolone derivative having thefollowing formula:

R and R² are independently H, OH, F, Cl, Br, I, C₁-C₆ alkyl, aryl or(CH₂)_(n)-aryl;

-   R¹ is H, OH, F, Cl, Br, I, C₁-C₆ alkyl, C₃-C₆ cycloalkyl,    CH═CH-aryl, C≡C-aryl, (CHR³)_(n)-aryl, NR³—C₁-C₆ alkyl,    NR³-cycloalkyl, NR³—(CHR³)_(n)-aryl, (CHR³)_(n)—NR³-aryl,    (CHR³)_(n)—NR³-alkyl, (CHR³)_(n)—NR³-cycloalkyl, (CHR³)_(n)—O-aryl,    (CHR³)_(n)—O-alkyl, (CHR³)_(n)-O-cycloalkyl, O—(CHR³)_(n)-aryl,    S—(CHR³)_(n)-aryl, or CO-aryl, wherein n is 0, 1, or 2 and alkyl,    cycloalkyl or aryl is optionally substituted with F, Cl, Br, I, CN,    CO₂H, CO₂R³, NO₂, CF₃, substituted or unsubstituted C₁-C₆ alkyl,    substituted or unsubstituted cycloalkyl, substituted or    unsubstituted aryl, OCF₃, OR³, OSO₂-aryl, substituted or    unsubstituted amine, NHCOR³, NHSO₂R³, CONHR³, or SO₂NHR³; and-   R³ is H, or substituted or unsubstituted C₁-C₆alkyl, substituted or    unsubstituted aryl.

Preferred groups represented by R¹ include —CH₃, Br,

It is yet another object of the present invention to provide ananti-thrombotic morpholino-substituted benzopyranone derivative havingthe following formula:

-   R is H, OH, F, Cl, Br, I, C₁-C₆ alkyl, aryl or (CH₂)_(n)-aryl;-   R¹ and R² are independently H, OH, F, Cl, Br, I, C₁-C₆ alkyl, C₃-C₆    cycloalkyl, CH═CH-aryl, C≡C-aryl, (CHR³)-aryl, NR³—C₁-C₆ alkyl,    NR³-cycloalkyl, NR³—(CHR³)_(n)-aryl, (CHR³)_(n)—NR³-aryl,    (CHR³)_(n)—NR³-alkyl, (CHR³)_(n)—NR³-cycloalkyl, (CHR³)_(n)—O-aryl,    (CHR³)_(n)—O-alkyl, (CHR³)_(n)—O-cycloalkyl, O—(CHR³)_(n)-aryl,    S—(CHR³)_(n)-aryl, or CO-aryl, wherein n is 0, 1, or 2 and alkyl,    cycloalkyl or aryl is optionally substituted with F, Cl, Br, I, CN,    CO₂H, CO₂R³, NO₂, CF₃, substituted or unsubstituted C₁-C₆ alkyl,    substituted or unsubstituted cycloalkyl, substituted or    unsubstituted aryl, OCF₃, OR³, OSO₂-aryl, substituted or    unsubstituted amine, NHCOR³, NHSO₂R³, CONHR³, or SO₂NHR³; and-   R³ is H, or substituted or unsubstituted C₁-C₆alkyl, substituted or    unsubstituted aryl.

Preferred groups represented by R¹ include —CH₃, Br,

Preferred compounds of the morpholino-substituted pyridopyrimidinederivatives are shown in Table I.

TABLE I TGX STRUCTURE TGX-167B

TGX-137

TGX-126

TGX-174

TGX-101

TGX-170

TGX-123

TGX-176

TGX-161

TGX-131

TGX-130

TGX-168

TGX-163

TGX-141

TGX-139

TGX-108

TGX-107

TGX-040

TGX-162

TGX-142

TGX-124

TGX-179

TGX-087

TGX-169

TGX-147

TGX-093

TGX-083

TGX-112

TGX-100

TGX-098

TGX-096

TGX-095

TGX-091

TGX-140

TGX-120

TGX-148

TGX-110

TGX-097

TGX-069

TGX-041

TGX-037

TGX-025

TGX-066

TGX-109

TGX-153

TGX-024

TGX-033

TGX-026

TGX-064

TGX-089

TGX-183

TGX-186

TGX-177

TGX-185

Preferred compounds of the morpholino-substituted quinolone derivativesare shown in Table II.

TABLE II TGX STRUCTURE TGX-155

TGX-127

TGX-115

TGX-121

TGX-111

TGX-084

TGX-180

TGX-143

TGX-113

TGX-149

TGX-152

TGX-151

TGX-171

TGX-099

TGX-106

TGX-057

TGX-070

TGX-077

TGX-071

TGX-086

TGX-078

TGX-074

TGX-138

Preferred compounds of the morpholino-substituted benzopyranonederivatives are shown in Table III.

TABLE III TGX STRUCTURE TGX-134

TGX-102

TGX-90

TGX-135

TGX-173

TGX-165

TGX-146

TGX-132

TGX-103

TGX-136

TGX-160

TGX-145

TGX-144

TGX-158

TGX-157

TGX-117

TGX-159

TGX-154

TGX-118

TGX-125

TGX-129

TGX-182

TGX-184

TGX-166

It is another object of the present invention to provide a method forinhibiting PI 3-kinase in a patient, comprising administering to thepatient an amount of one of the compounds of the present invention,wherein the amount is effective in inhibiting the phosphoinositide3-kinase in the patient.

It is still another object of the present invention to provide a methodfor preventing or treating cardiovascular disease, such as coronaryartery occlusion, stroke, acute coronary syndrome, acute myocardialinfarction, restenosis, atherosclerosis, and unstable angina, byadministering an effective amount of one of the compounds of the presentinvention to a patient in need thereof. Similarly, the present inventioncontemplates preventing or treating respiratory disease, for example,asthma, chronic obstructive pulmonary disease, and brochitis, or acancer condition, such as a glioma, prostate cancer, small cell lungcancer, and breast cancer, by administering an effective amount of oneof the compounds of the present invention to a patient in need thereof.

Another object of the present invention relates to a method forpreventing or treating disease linked to disordered white blood cellfunction, e.g., autoimmune disease and inflammatory disease, byadministering, to a patient in need thereof, an effective amount of oneof the compounds of the present invention.

Advantageously, in the present methods for preventing or treating adisease condition, the effective amount of one of the present compoundsis administered in the form of a dose. In preferred embodiments, thedose is preferably in the form of a tablet (e.g., a tablet formulatedfor oral, sublingual, and buccal administration), capsule (e.g., acapsule containing powder, liquid, or a controlled-release formulation),intravenous formulation, intranasal formulation, formulation formuscular injection, syrup, suppository, aerosol, buccal formulation,transdermal formulation, or pessary. Preferably, the dose contains fromabout 5 to about 500 mg of the compound, and more preferably containsfrom about 25 to about 300 mg of the compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a bar graph illustrating the effect, in a flow-basedreconstitution assay, of various concentrations of TGX-40 on theadhesion of platelets to vWf-coated glass microslides;

FIG. 2 shows photographs and a bar graph illustrating the effect, in awhole-blood flow assay, of various concentrations of TGX-40 on theadhesion of platelets to vWf-coated glass microslides;

FIG. 3 shows a bar graph illustrating the effect, in an animal model ofarterial occlusion, of two concentrations of TGX-40 on the stabilizationof blood flow in rats producing regular cyclic flow reductions (CFRs);and

FIG. 4 shows a bar graph illustrating the effect, in a whole-blood flowassay, of various concentrations of TGX-84 on the platelet thrombusformation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the context of this description, the term “alkyl” refers to straightor branched saturated aliphatic hydrocarbon radical. Preferably, thealkyl group has 1 to 6 carbons and optionally substituted with one ormore groups selected from halogen such as F, Cl, Br or I; CN; CO₂R₃;NO₂; CF₃; substituted or unsubstituted C₁-C₆ alkyl; substituted orunsubstituted C₃-C₆ cycloalkyl; substituted or unsubstituted aryl; OCF₃,OR₃, substituted or unsubstituted amine; NHCOR₃; NHSO₂R₃; CONHR₃; orSO₂NHR₃, wherein R₃ is H, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted aryl.

The term “cycloalkyl” refers to non-heterocyclic (i.e., carbocyclic) orheterocyclic ring. Exemplary of non-heterocyclic ring in this regard issubstituted or unsubstituted cyclopropane, cyclobutane, cyclopentane,cyclohexane, cyclohexadione, cyclopentanedione, quinone and the like.Suitable heterocycloalkyl groups include substituted or unsubstitutedpyrrolidine, piperidine, piperazine, 2-piperidone, azacyclohexan-2-oneand morpholine groups. The cycloalkyl group is optionally substituted atone or more positions with halogen such as F, Cl, Br or I; CN; CO₂R₃;NO₂; CF₃, substituted or unsubstituted C₁-C₆ alkyl; substituted orunsubstituted C₃-C₆ cycloalkyl; substituted or unsubstituted aryl; OCF₃,OR₃, substituted or unsubstituted amine; NHCOR₃; NHSO₂R₃; CONHR₃; orSO₂NHR₃, wherein R₃ is H, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted aryl.

The term “aryl” refers to an aromatic or heteroaromatic rings. Examplesof an aryl group are pyrrolidine, thiophene, pyrrole, pyrazole,imidazole, 1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole,isothiazole, furan, 1,2,3-oxadiazole, 1,2,4-oxadiazole,1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3,4-oxatriazole,1,2,3,5-oxatriazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole,1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3,4-thiatriazole,1,2,3,5-thiatriazole, tetrazole, benzene, pyridine, pyridazine,pyrimidine, pyrazine, triazine, indene, naphthalene, indole, isoindole,indolizine, benzofuran, benzothiophene, indazole, benzimidazole,benzthiazole, purine, quinolizine, quinoline, isoquinoline, cinnoline,phthalazine, quinazoline, quinoxaline, naphthyridine, pteridine,fluorene, carbazole, carboline, acridine, phenazine, and anthracene. Thearyl group is optionally substituted at one or more positions withhalogen such as F, Cl, Br or I; CN; CO₂R₃; NO₂; CF₃, substituted orunsubstituted C₁-C₆ alkyl; substituted or unsubstituted C₃-C₆cycloalkyl; substituted or unsubstituted aryl; OCF₃, OR₃, substituted orunsubstituted amine; NHCOR₃; NHSO₂R₃; CONHR₃; or SO₂NHR₃, wherein R₃ isH, substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted aryl.

The morpholino-substituted compounds of the present invention have beenfound to inhibit the lipid signalling enzyme PI 3-kinase, whichregulates platelet-adhesion processes under blood-flow conditions, andtherefore to display anti-thrombotic activity, as well as otherpharmacological properties elaborated below. PI 3-kinase generates3-phosphorylated PI second messengers, includingphosphatidylinositol-3-phosphate (PI(3)P),phosphatidylinositol-3,4-bisphosphate (PI(3,4)P₂), andphosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P₃). These secondmessengers are thought to regulate a diverse range of cellularphenomena, including glucose transport, apoptosis prevention, vesiculartrafficking, cell growth, and cytoskeletal reorganization.

To the inventors' knowledge, there are no published reports on theeffects of PI 3-kinase inhibitors on platelet adhesion underpathophysiologically relevant flow conditions. Nevertheless, it has beendiscovered that PI 3-kinase plays a critical role in regulating plateletadhesion, particularly under conditions of physiological flow. Thus,treatment of platelets with the compounds of the present inventioninhibit the formation of the phosphorylated lipid products of PI3-kinase, PI(3)P, PI(3,4)P₂, and PI(3,4,5)P₃, effecting a markedreduction in platelet adhesion to a vWf matrix under flow conditions.This reduction in platelet adhesion is associated with abnormal plateletspreading and thrombus formation. Because shear-dependent plateletadhesion and activation is important in arterial thrombus formation, PI3-kinase is an important target for therapeutic intervention incardiovascular diseases generally.

These inhibitors of PI 3-kinase also have potential therapeutic uses ina variety of other disease states. For example, PI 3-kinase plays animportant role in promoting smooth muscle proliferation in the vasculartree, i.e., vascular smooth muscle cells (Thyberg, 1998, EuropeanJournal of Cell Biology 76(1):33-42), and in the lungs (airway smoothmuscle cells). Krymskaya et al., 1999, American Journal of Physiology277:65-78. Excessive proliferation of vascular smooth muscle cells playsan important role in the formation of atherosclerotic plaques and in thedevelopment of neointimal hyperplasia following invasive vascularprocedures. Scwartz et al., 1984, Progress in Cardiovascular Disease26:355-372; Clowes et al., 1978, Laboratory Investigations 39:141-150.Moreover, excessive proliferation of airway smooth muscle cells leads tothe development of COPD in the setting of asthma and chronic bronchitis.Inhibitors of PI 3-kinase therefore may be used to prevent vascularrestenosis, atherosclerosis, and COPD.

PI 3-kinase also plays an important role in regulating tumor cells andin the propensity of these cells to undergo apoptosis growth. Sellers etal., 1999, The Journal of Clinical Investigation 104:1655-1661.Additionally, uncontrolled regulation of the PI 3 kinase lipid productsPI(3,4,5)P₃ and PI(3,4)P₂ by the lipid phosphatase PTEN plays animportant role in progression of a number of malignant tumors in humans.Leevers et al., 1999, Current Opinion in Cell Biology 11:219-225.Therefore, inhibitors of PI 3-kinase may be used to treat neoplasms inhumans.

PI 3-kinase also plays an important role in leukocyte function (Fulleret al., 1999, The Journal of Immunology 162(11):6337-6340; Eder et al.,1998, The Journal of Biological Chemistry 273(43):28025-31) andlymphocyte function (Vicente-Manzanares et al., 1999, The Journal ofImmunology 163(7):4001-4012). For example, leukocyte adhesion toinflamed endothelium involves activation of endogenous leukocyteintegrins by a PI 3-kinase-dependent signaling process. Furthermore,oxidative burst (Nishioka et al., 1998, FEBS Letters 441(1):63-66) andcytoskeletal reorganization (Kirsch et al., 1999, Proceedings NationalAcademy of Sciences 96(11):6211-6216) in neutrophils appears to involvePI 3-kinase signaling. Thus, inhibitors of PI 3-kinase may be useful inreducing leukocyte adhesion and activation at sites of inflammation andtherefore may be used to treat acute and/or chronic inflammatorydisorders. PI 3-kinase also plays an important role in lymphocyteproliferation and activation. Fruman et al., 1999, Science 283(5400):393-397. Given the important role of lymphocytes in auto-immunediseases, inhibitors of PI 3-kinase may be used in the treatment of suchdisorders.

The invention is further described by reference to the followingexamples, which are set forth by way of illustration only. Nothing inthese examples should be taken as a limitation upon the overall scope ofthe invention.

EXAMPLE 1 Preparation of Morpholino-Substituted PyridopyrimidineDerivatives

The morpholino-substituted pyridopyrimidine compounds of the presentinvention may be prepared using a common synthetic scheme, illustratedin this example, differing only in the starting 2-amino pyridine.Specifically, an appropriately substituted 2-amino pyridine is treatedwith diethylmalonate to yield a hydroxy-substituted pyridopyrimidine.The hydroxy-substituted pyridopyrimidine is subsequently reacted withphosphorus oxychloride to give a chloro-substituted pyridopyrimidine.Finally, the chloro-substituted pyridopyrimidine is reacted withmorpholine to yield the morpholino-substituted pyridopyrimidine.

The present morpholino-substituted pyridopyrimidine derivatives wereprepared according to the following general synthetic scheme:

The starting substituted 2-amino pyridine (compound 1) was2-amino-3-methyl pyridine for TGX-25 (compound 2), 2-amino-3-phenylpyridine for TGX-37 (compound 3), 2-amino-3-phenyl-5-methylpyridine forTGX-41 (compound 4), and 2-amino-3-benzyl pyridine for TGX-40 (compound5).

2-amino-3-phenyl pyridine was prepared as follows: 3-phenyl pyridine(300 mg, 2 mmol) was dissolved in para-xylene (6 ml), and sodamide (84mg, 2.1 mmol) was then added. The reaction mixture was heated to refluxtemperature for 8 hours. The reaction mixture was cooled, poured ontoice/water (25 ml), and extracted with dichloromethane. The organicextracts were washed with water and brine, and dried over anhydroussodium sulphate. The sodium sulphate was removed by filtration, and thefiltrate was evaporated to dryness and recrystallized from a mixture ofdiethyl ether and petroleum ether to provide 2-amino-5-phenyl pyridine(95 mg). The mother liquors from the crystallization were evaporated todryness and subjected to purification by column chromatography (silica),thereby eluting the solvent ethyl acetate:petroleum ether (30:70). Thedesired product, 2-amino-3-phenyl pyridine, was obtained as a fineyellow powder (15 mg).

2-amino-3-phenyl-5-methylpyridine was prepared as follows:2-amino-5-picoline (10.8 g, 0.1 M) was dissolved in glacial acetic acid(200 ml), and N-bromosuccinamide (20 g, 0.11 M) was added. The reactionmixture was stirred at room temperature for 17 hours. The reactionmixture was poured onto ice/water and the solid removed by filtration.The filtrate was basified with solid sodium hydroxide, and the resultingprecipitate was isolated by filtration (12.8 g). The product,2-amino-3-bromo-5-methylpyridine (3.7 g, 20 mmol), was dissolved inanhydrous DMSO (100 ml) under an atmosphere of nitrogen. Phenylboronicacid (2.66 g, 22 mmol) was added, followed by the addition of potassiumcarbonate (9.66 g, 70 mmol) and bis(triphenylphosphine)-palladium(II)chloride (426 mg, 0.6 mmol). The reaction mixture was heated to 80° C.with stirring for 15 hours. The reaction was cooled, poured intoice/water, and the crude product was collected by filtration. Theresultant material was treated with 1 M aqueous hydrochloric acid (200ml), stirred for 10 minutes, and filtered to remove insoluble residues.The filtrate was basified with solid sodium hydroxide, and the resultantyellow precipitate was filtered and dried to provide the product,2-amino-3-phenyl-5-methylpyridine, as a pale yellow solid (2.25 g).

2-amino-3-benzyl pyridine was prepared from 3-benzyl pyridine asdescribed in Kelly et al., 1990, The Journal of the American ChemicalSociety 112:8024 (1990).

TGX-40 was prepared as follows: 2-amino-3-benzyl-pyridine (5.4 g) wastreated with diethylmalonate (12 g) at 190-200° C. for 40 min. Theexcess of diethylmalonate was evaporated with a stream of nitrogen gasat the same temperature. The resulting solid was triturated three timeswith diethylether and dried in vacuo (2.4 g, 28%). Thishydroxypyrimidine derivative (528 mg) was then treated with an excess ofPOCl₃ (6 ml) and refluxed for 45 min. The reaction mixture was broughtto room temperature and poured onto ice. The resulting precipitate wasfiltered and dried (341 mg, 59%). The crude chloroderivative (191 mg)was dissolved in ethanol (10 ml) containing morpholine (1 ml) andrefluxed for 4 hrs. The reaction mixture was brought to room temperatureand concentrated in vacuo. The residue was treated with aqueousbicarbonate and the resulting precipitate was then filtered and dried(151 mg, 78%).

TGX-101 was prepared as follows:

A mixture of a bromo derivative (compound 1) (324 mg, 1 mmol),4-aminophenol (compound 2) (110 mg, 1 mmol), potassium t-butoxide (225mg, 2 mmol), and PdCl₂ (dppf) (35 mg, 0.05 mmol) in THF was stirred atrefluxing temperature for 20 hours over a nitrogen atmosphere. Thereaction mixture was cooled and concentrated in vacuo. The resultingresidue was diluted with water to give a dark green precipitate, whichwas filtered and dried. The solid was further purified by trituratingwith diethyl ether (two times) and dichloromethane (two times)successively, to give the required product (compound 3) (140 mg).

¹H NMR (300 MHz, DMSO) for TGX-101: δ 9.37 (s, 1H, —OH), 7.96 (s, 1H),7.79 (s, 1H, —NH), 7.13 (d, J=8.7 Hz, 2H), 6.80 (d, J=8.7 Hz, 2H), 6.66(d, J=1.8 Hz, 1H), 5.58 (s, 1H), 3.65 (br s, 8H), 2.16 (s, 3H).

By means of the above procedure, TGX-107 was prepared from the bromoderivative (compound 1) and 4-chloroaniline, TGX-108 was prepared bycoupling compound 1 with 4-chlorobenzylamine, TGX-109 was prepared bycoupling compound 1 with para-cresol, TGX-112 was prepared by couplingcompound 1 with 4-pyridylamine, TGX-120 was prepared by couplingcompound 1 with 4-aminopyridine. In a similar manner, TGX-123 andTGX-124 and TGX-126 and TGX-130 were prepared by coupling compound 1with the appropriate substituted amine.

EXAMPLE 2 Preparation of 8-substituted2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-ones

8-Substituted 2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-ones wereprepared according to general procedure shown below. In brief,8-benzyloxy-2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-one (TGX-131) wasdebenzylated by treatment with trifluoromethane sulfonic anhydride andthe resultant 8-hydroxy-2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-onewas derivatised by copper-promoted arylation using arylboronic acidsadapting the method of Evans et al., 1998. Tetrahedron Lett.39:2937-2940 (Example 2A) or base catalysed alkylation using arylmethylhalides (Example 2B).

EXAMPLE 2A 2-Morpholinyl-8-phenoxy-4H-pyrido[1,2-a]pyrimidin-4-one(TGX-141) 8-Hydroxy-2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-one

A solution of 8-benzyloxy-2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-one(0.97 g, 2.9 mmol) in dichloromethane (50 ml) under nitrogen was treateddropwise with trifluoromethanesulfonic anhydride (1 ml, 6.0 mmol) andthe mixture was stirred at RT overnight. Methanol (20 ml) was added andthe solution stirred for a further 1 h, then the solution was evaporatedto dryness. The residue was taken up in ethyl acetate, washed withbrine, dried and then eluted through a silica column using a gradient of0-5% methanol in ethyl acetate. The product was obtained as a brownpowder (0.35 g)

2-Morpholinyl-8-phenoxy-4H-pyrido[1,2-a]pyrimidin-4-one (TGX-141)

8-Hydroxy-2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-one (0.2 g, 0.81mmol), phenylboronic acid (0.29 g, 2.4 mmol), and copper acetate (0.20g, 1.6 mmol) were suspended in dichloromethane and treated withtriethylamine (0.23 ml, 1.6 mmol) and the mixture was stirred at RT for4 days. The product was adsorbed onto silica and eluted through a silicacolumn with ethyl acetate to yield a pale tan solid (0.026 g).

¹H-NMR (CDCl₃, 300 MHz): δ 3.45 (t, 4H, J=5 Hz), 3.63 (t, 4H, J=5 Hz),5.59 (s, 1H), 6.8 (t, 1H, J=8 Hz), 7.40 (t, 2H, J=8.7 Hz), 7.45-7.55 (m,3H), 8.18 (dd, 1H, J=9.0 Hz, 2 Hz).

In a similar manner but utilizing the appropriate arylboronic acid wasalso prepared:

-   8-(4-Fluoro-3-methylphenyl)oxy-4H-pyrido[1,2-a]pyrimidin-4-one    (TGX-168) and-   8-(2-methylphenyl)oxy-4H-pyrido[1,2-a]pyrimidin-4-one (TGX-182)

EXAMPLE 2B8-(2-chloropheinyl)methoxy-2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-one(TGX-177)

8-Hydroxy-2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-one (59 mg, 0.24mmol) was dissolved in acetonitrile (10 ml) and then treated withanhydrous potassium carbonate (197 mg, 1.4 mmol) followed by2-chlorobenzylbromide (46 mg, 0.29 mmol) and the mixture was stirred at80° C. overnight. Upon cooling the mixture was adsorbed directly ontosilica, then eluted through a silica column using ethyl acetate. Thepurified product was obtained as a tan solid (34 mg).

¹H-NMR (CDCl₃, 300 MHz): δ 3.70 (t, 4H, J=5 Hz), 3.80 (t, 4H, J=5 Hz),5.34 (s, 2H), 5.66 (s, 1H), 6.8 (t, 1H, J=8 Hz), 7.00 (d, 1H, J=8 Hz),7.30 (m, 2H), 7.4 (m, 1H), 7.65 (m, 1H), 8.58 (d, 1H, J=8 Hz).

In a similar fashion but utilizing the appropriate arylmethyl halidewere prepared:

-   8-(2-pyridinylmethyl)oxy-2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-one    (TGX-148);-   8-(3-pyridinylmethyl)oxy-2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-one    (TGX-140);-   8-(4-pyridinymethyl)oxy-2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-one    (TGX-185);-   8-(3-chlorophenyl)methoxy-2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-one    (TGX-176);-   8-(4-bromophenyl)methoxy-2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-one    (TGX-175);-   8-(4-t-butylphenyl)methoxy-2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-one    (TGX-169); and-   8-(3-methoxyphenyl)methoxy-2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-one    (TGX-163).

EXAMPLE 2C6-methyl-8-phenylaminomethyl-2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-one(TGX-183)

TGX-183 was synthesised according to the following scheme. The keyreactions involved in this synthetic sequence are a palladium catalysedvinylation and one step cleavage of the alkene functionality to analdehyde group.

Reagents: a). 4-vinylpyridine, Cs₂CO₃, PdCl₂(dppf), DMF, 80° C., 16 hrs,b). CTAP, CH₂Cl₂, 2 hrs. RT c). i. NaBH₄, methanol, 0.5 hrs, RT, ii.methanesuphonyl chloride, Et₃N, CH₂Cl₂, 0° C. then aniline, reflux, 4hrs.The preparation of aldehyde 3 as follows:

A mixture of bromo compound 1 (324 mg, 1 mmol), 4-vinylpyridine (0.5mL), CsCO₃ (0.98 g, 3 mmol), PdCl₂(dppf) (35 mg) in DMF (10 mL) washeated at 80° C. for 16 hours over a nitrogen atmosphere. The reactionmixture was brought to room temperature and poured onto ice. Theresulting precipitate was filtered, dried in vacuo and taken to the nextoxidation reaction without further purification. ¹H NMR (300 MHz,CDCl₃): δ 8.76 (s, 1H), 8.63 (d, J=4.73 Hz, 2H), 7.95 (d, J=16.63 Hz,1H), 7.80 (d, J=1.98 Hz, 1H), 7.39 (d, J=6.10 Hz, 2H), 7.19 (d, J=16.48Hz, 1H) 5.66 (s, 1H), 4.56 (s, 2H), 3.82 (m, 4H), 3.68 (m, 4H), 2.39 (s,3H).

The crude product 2 obtained from the above reaction, was dissolved indichloromethane (30 mL) to which was added cetyltrimethylammoniumpermanganate (Bhushan, V., et al Synthesis, 431, 1984) (0.5 g). Thereaction mixture was stirred for 5 hours at room temperature. Thereaction mixture was concentrated in vacuo to half of its originalvolume and adsorbed on silica gel. The required product 3 was isolatedby short path column chromatography (silica gel, ethylacetate) as ayellow solid (158 mg, 58%). ¹H NMR (300 MHz, DMSO): δ 10.7 (s, 1H), 8.84(s, 1H), 8.11 (s, 1H), 5.67 (s, 1H), 3.66 (br s, 8H), 2.35 (s, 3H).

The preparation of TGX-183 (4) as follows:

The yellow-coloured aldehyde 3 (158 mg) was suspended in methanol (5 mL)and reacted with sodium borohydride (20 mg) at room temperature.Stirring was continued until the reaction colour became white. Thereaction mixture was concentrated in vacuo and diluted with water. Theresulting white precipitate was filtered and dried to give the requiredproduct (150 mg) which was taken to next synthetic step without furtherpurification. ¹H NMR (300 MHz, CDCl₃): δ 8.67 (s, 1H), 7.48 (s, 1H),5.63 (s, 1H), 4.84 (br s, 2H), 3.80 (m, 4H), 3.60 (m, 4H), 2.34 (s, 3H).

The crude product (150 mg) which obtained from the previous reaction wassuspended in dichloromethane (10 mL) to which was added triethylamine(0.14 mL, 1 mmol) followed by methanesulphonyl chloride (0.078 mL, 1mmol) at ice-cold temperature. After 15 minutes, aniline (0.18 ml, 2mmol) was added and refluxed for 4 hours. The reaction mixture wascooled and diluted with dichloromethane (50 ml). The dichloromethanelayer was washed with water, brine and dried over sodium sulphate. Afterevaporation of the solvent in vacuo, the residue was purified by columnchromatography (silica gel, ethyl acetate) to give the pure anilinederivative 4 (TGX-183) (120 mg). ¹H NMR (300 MHz, CDCl₃): δ 8.65 (s,1H), 7.52 (s, 1H), 7.18 (m, 2H), 6.74 (br t, 1H), 6.61 (br d, 2H), 5.64(s, 1H), 4.56 (s, 2H), 3.79 (m, 4H), 3.63 (m, 4H), 2.28 (s, 3H).

TGX-183:

¹H NMR (300 MHz, DMSO): δ 8.52 (s, 1H), 7.52 (s, 1H), 7.06 (d, J=7.0 Hz,1H), 7.03 (d, J=7.0 Hz, 1H), 6.55-6.50 (m, 3H), 6.20 (t, J=5.8 Hz, 1H,—NH), 5.62 (s, 1H), 4.44 (d, J=5.8 Hz, 2H), 3.66-3.59 (m, 8H), 2.23 (s,3H).

6-methyl-8-(2-methyl-4-fluorophenyl)aminomethyl-2-morpholinyl-4H-pyrido[1,2-a]pyrimidin-4-one(TGX-186)

(5) was prepared according to the procedure described for TGX-183 (4)except 4-fluoro-2-methylaniline was used instead of aniline during thelast step of the synthetic sequence.

¹H NMR (300 MHz, DMSO): δ 8.52 (s, 1H), 7.47 (s, 1H), 6.87 (dd, J=9.5,3.0 Hz, 1H), 6.72 (m, 1H), 6.23 (dd, J=9.0, 4.9 Hz, 1H), 5.63 (s, 1H),5.52 (t, J=5.8 Hz, 1H, —NH), 4.49 (d, J=6.1 Hz, 2H), 3.67-3.60 (m, 8H),2.23 (s, 3H), 2.17 (s, 3H).

EXAMPLE 3 Preparation of Morpholino-Substituted Quinolone Derivatives

The morpholino-substituted quinolone compounds of the present inventionwere prepared according to the following general synthetic scheme:

TGX-57 (compound 14), TGX-84 (compound 15), TGX-115 (compound 16) andTGX-155 (compound 17) were prepared by adapting the procedure of Huanget al., 1989, Synthesis 317, starting with the appropriately substitutedaniline and Meldrum's acid derivative (compound 1) (Huang et al., 1986,Synthesis 967). Anilines (compound 4) and (compound 5) in turn weresynthesized by reaction of 2-chloronitrobenzene with o-cresol or4-fluoro-o-cresol to yield the corresponding nitro compounds followed byPd catalyzed hydrogenation.

Substitution of Meldrum's acid derivative (compound 1) with2-benzylaniline (compound 2) (or 2-phenoxyanilines, compounds 3-5)yielded intermediate compound 6 (or compounds 7-9) which after reactionwith morpholine resulted in compound 10 (or compounds 11-13). Finally,the required quinolinone skeleton was constructed by refluxing compound10 (or compounds 11-13) in diphenyl ether for 15 minutes.

Anilines (compounds 4 and 5) were prepared as follows: A mixture of2-Chloronitrobenzene (5.68 g, 36 mmol), o-cresol (or 4-fluoro-o-cresol)(40 mmol) and potassium carbonate (14.9 g, 108 mmol) in DMSO (120 mL)was stirred at 80° C. for 18 h. Water (60 mL) was added and the reactionmixture extracted with ethyl acetate (3×200 mL). The combined organicextracts were sequentially washed with 1M sodium hydroxide (3×100 mL)and aqueous sodium chloride (100 mL), dried (sodium sulphate) andevaporated under reduced pressure to yield the correspondingnitrocompounds (95-100%). Pd/C catalysed hydrogenation of nitrocompoundsin ethanol at ambient temperature for 7 h yielded the required anilines.The catalyst was filtered and the resultant filtrate evaporated underreduced pressure to yield aniline (compound 4) (or compound 5) as abrown oil (90-95%). The crude anilines (compounds 4-5) were used withoutfurther purification in the subsequent reaction with Meldrum's acidderivative (compound 1).

5-[Anilino(methylthio)methylene]-2,2-dimethyl-4,6-dioxo-1,3-dioxanes(compounds 6-9) were prepared as follows: A mixture of5-[bis(methylthio)methylene]-2,2-dimethyl-4,6-dioxo-1,3-dioxane(compound 1) (2.48 g, 10 nmol), 2-substituted aniline (compound 2) (orcompounds 3-5) (10 mmol) in ethanol (25 mL) was heated at 140° C. for4.5 h. Evaporation of the solvent under reduced pressure yielded a crudeyellow oil which, after purification by flash chromatography, usingpetroleum ether/ethyl acetate (9:1 and then 3:1) as eluent, yieldedcompound 6 (or compounds 7-9) (68-77%).

¹H NMR (300 MHz; CDCl₃) for5-[2-benzylanilino(methylthio)methylene]-2,2-dimethyl-4,6-dioxo-1,3-dioxane(compound 6): δ 1.73 (6H, s, CH₃), 1.89 (3H, s, CH₃), 4.04 (2H, s, CH₂),7.08-7.41 (9H, m, CHAr). ¹H NMR (300 MHz; CDCl₃) for5-[Methylthio-(2-phenoxyanilino)methylene]-2,2-dimethyl-4,6-dioxo-1,3-dioxane(compound 7): ¹H NMR (300 MHz; CDCl₃): δ 1.62 (6H, s, CH₃), 2.20 (3H, s,CH₃), 6.90-7.72 (9H, m, CHAr). ¹H NMR (300 MHz; CDCl₃) for5-[2-(2′-Methylphenoxy)anilino(methylthio)-methylene]-2,2-dimethyl-4,6-dioxo-1,3-dioxane(compound 8): δ 1.70 (6H, s, CH₃), 2.25 (3H, s, CH₃), 2.31 (3H, s, CH₃),6.80-7.44 (8H, m, CHAr). ¹H NMR (300 MHz; CDCl₃) for5-[2-(4′-Fluoro-2′-methylphenoxy)anilino(methylthio)methylene]-2,2-dimethyl-4,6-dioxo-1,3-dioxane(compound 9): δ 1.72 (6H, s, CH₃), 2.22 (3H, s, CH₃), 2.33 (3H, s, CH₃),6.72-7.44 (7H, m, CHAr), 7.8 (1H, s, NH).

5-[Anilino(morpholino)methylene]-2,2-dimethyl-4,6-dioxo-1,3-dioxanes(compounds 10-13) were prepared as follows: A mixture of5-[anilino(methylthio)-methylene]-2,2-dimethyl-4,6-dioxo-1,3-dioxane(compound 6) (or compounds 7-9) (18 mmol) and morpholine (3.15 mL, 36mmol) in tetrahydrofuran (100 mL) was heated at reflux temperatureovernight. The solvent was evaporated and the crude yellow solid waswashed with ether to yield compound 10 (or compounds 11-13) (90-95%) asa white solid. ¹H NMR (300 MHz; CDCl₃) for5-[2-Benzylanilino(morpholino)methylene]-2,2-dimethyl-4,6-dioxo-1,3-dioxane(compound 10): δ 2.04 (6H, s, CH₃), 3.39 (4H, t, J4.9 Hz, CH₂), 3.70(4H, t, J4.9 Hz, CH₂), 6.55 (1H, d, J7.5 Hz, CHAr), 6.95 (1H, td, J7.5,1.2 Hz, CHAr), 7.08-7.23 (7H, m, CHAr). ¹H NMR (300 MHz; CDCl₃) for5-[Morpholino-(2-phenoxyanilino)methylene]-2,2-dimethyl-4,6-dioxo-1,3-dioxane(compound 11): δ 1.60 (6H, s, CH₃), 3.31 (4H, t, J4.7 Hz, CH₂), 3.71(4H, t, J4.7 Hz, CH₂), 6.92-7.35 (9H, m, CHAr). ¹H NMR (300 MHz; CDCl₃)for5-[2-(2′-Methylphenoxy)anilino(morpholino)-methylene]-2,2-dimethyl-4,6-dioxo-1,3-dioxane(compound 12): δ 1.67 (6H, s, CH₃), 2.19 (3H, s, CH₃), 3.32 (4H, t, J4.6Hz, CH₂), 3.74 (4H, t, J4.6 Hz, CH₂), 6.73 (1H, dd, J=8.0, 1.5 Hz,CHAr), 6.90 (1H, dd, J8.0, 1.5 Hz, CHAr), 7.08-7.24 (6H, m, CHAr), 9.51(1H, s, NH). ¹H NMR (400 MHz; CDCl₃) for5-[2-(4′-Fluoro-2′-methylphenoxy)anilino-(morpholino)methylene]-2,2-dimethyl-4,6-dioxo-1,3-dioxane(compound 13): δ 1.68 (6H, s, CH₃), 2.17 (3H, s, CH₃), 3.33 (4H, t, J4.6Hz, CH₂), 3.74 (4H, t, J4.6 Hz, CH₂), 6.66 (1H, dd, J8.0, 1.2 Hz, CHAr),6.89-7.29 (5H, m, CHAr), 7.41 (1H, t, J8.0 Hz, CHAr), 9.47 (1H, s, NH).

2-Morpholino-4-quinolones (compound 14-17; TGX-57, TGX-84, TGX-115 andTGX-155) were prepared as follows:5-[Anilino(morpholino)methylene]-2,2-dimethyl-4,6-dioxo-1,3-dioxane(compound 10) (or compounds 11-13) was heated in diphenyl ether (3-4 mL)at 240° C. for 15 minutes. The reaction mixture was cooled to roomtemperature and petroleum ether (bp 60-90° C., 30 mL) added to yield thecrude compound which after purification by flash chromatography, usingpetroleum ether/ethyl acetate (1:1) and then ethyl acetate/methanol(9:1) as eluent, yielded compound 14 (or compounds 14-17) (40-50%). ¹HNMR (400 MHz; CDCl₃) for 8-Benzyl-2-morpholino-4-quinolone (compound 14;TGX-57): δ 3.11 (4H, t, J4.6 Hz, CH₂), 3.58 (4H, t, J4.6 Hz, CH₂), 4.44(2H, s, CH₂), 6.75 (1H, s, CH), 7.21-7.33 (6H, m, CHAr), 7.59 (1H, d,J7.3 Hz, CHAr), 7.78 (1H, d, J7.3 Hz, CHAr). ¹H NMR (400 MHz; CDCl₃) for2-Morpholino-8-phenoxy-4-quinolone (compound 15; TGX-84): δ 3.30 (4H, t,J5 Hz, CH₂), 3.82 (4H, br.s, CH₂), 5.80 (1H, s, CH), 6.98 (1H, d, J7.5Hz, CHAr), 7.09 (2H, d, J8.0 Hz, CHAr), 7.13 (1H, t, J8.0 Hz, CHAr),7.20 (1H, t, J7.5 Hz, CHAr), 7.40 (2H, t, J8.0 Hz, CHAr), 7.98 (1H, dd,J7.5, 1.2 Hz, CHAr). ¹H NMR (300 MHz; CDCl₃) for8-(2′-Methylphenoxy)-2-morpholino-4-quinolone (compound 16; TGX 115): δ2.24 (3H, s, CH₃), 3.33 (4H, t, J4.8 Hz, CH₂), 3.87 (4H, t, J4.8 Hz,CH₂), 5.80 (1H, s, CH), 7.01 (1H, d, J8.1 Hz, CHAr), 7.08 (1H, t, J8.0Hz, CHAr), 7.16-7.29 (3H, m, CHAr), 7.32 (1H, d, J8.0 Hz, CHAr), 7.92(1H, d, J 8.0 Hz, CHAr), 8.26 (1H, s, NH). ¹H NMR (300 MHz; CDCl₃) for8-(4′-Fluoro-2′-methylphenoxy)-2-morpholino-4-quinolone (compound 17;TGX 155): δ 2.20 (3H, s, CH₃), 3.35 (4H, t, J4.8 Hz, CH₂), 3.88 (4H, t,J4.8 Hz, CH₂), 5.80 (1H, s, CH), 6.65 (1H, d, J 8.1 Hz, CHAr), 6.95-7.10(4H, m, CHAr), 7.92 (1H, d, J8.1 Hz, CHAr), 8.23 (1H, s, NH).

With the appropriately 2-substituted aniline and with coupling with theMeldrum's acid derivative (compound 3), TGX-99, TGX-106, TGX-111,TGX-113, and TGX-121 were prepared as outlined above.

EXAMPLE 4 Preparation of Morpholino-Substituted BenzopyranoneDerivatives

8-(Substituted)-2-(4-morpholinyl)-4H-1-benzopyran-4-ones were preparedaccording to the following general procedure adapted from Morris et al.,1994, Synth. Commun, 24: 849-858. In brief, the lithium enolate ofacetyl morpholine is reacted with a substituted salicylate ester (1) toyield an intermediate salicylacetamide (2). Cyclodehydration of (2) withtrifluoromethanesulfonic anhydride yields the substituted morpholinosubstituted-benzopyranone (3).

Specific substituents in the 8-position of the product (3) wereintroduced either into the precursor (1) (Method A) or by elaboration of2-(4-morpholinyl)-8-trifluoromethanesulfonyloxy-4H-1-benzopyran-4-one(3, R═CF₃SO₃) (Methods B and C).

Method A

EXAMPLE A-1 2-morpholinyl-8-(phenylmethyl)-4H-1-benzopyran-4-one(TGX-90) 3-(phenylmethyl)salicylaldehyde

To a warm, stirred mixture of sodium hydroxide (8.0 g) in water (8.0 ml)was added a warmed solution of 2-hydroxydiphenylmethane (1), (4.9 g, 27mmol) in ethanol (4 ml) and the mixture heated to 65° C. Chloroform (4.1ml) was added down a water condensor and the resulting mixture began toreflux. After 1 h at reflux, the mixture was cooled in ice, acidified topH 2 with 1N HCl and extracted with ethyl acetate (3×30 ml). Thecombined extracts were dried (Na₂SO₄) and the solvent removed to yield adark brown gum. The product was eluted through a silica column, using0-10% ethyl acetate in petroleum spirit to yield a yellow oil (1.33 g,24%)

Methyl 3-(phenylmethyl)salicylate

According to the general method of Sharma et al., 2000, Synth. Commun.,30:397-405, a stirred solution of the 3-(phenylmethyl)salicylaldehyde(1.27 g, 6 mmol) in ethanol (16 ml) was treated dropwise with a solutionof silver nitrate (2.0 g, 12 mmol) in water (16 ml). A solution ofpotassium hydroxide (2.69 g, 48 mmol) was then added dropwise over 40minutes. The solution was allowed to stir at RT for 6 h. The mixture wasfiltered through a pad of celite, and the filter pad washed with water(2×10 ml). The filtrate was washed with diethyl ether (2×15 ml) and thenacidified with 1N HCl. The milky suspension was extracted with diethylether (2×30 ml), and the combined extracts were dried (Na₂SO₄) and thesolvent removed to yield 3-(phenylmethyl)salicylic acid as a tan solid(0.47 g, 34%). ¹H-NMR (CDCl₃, 400 MHz): δ 4.02 (s, 2H), 6.84 (t, 1H, J=8Hz), 7.19-7.32 (m, 6H), 7.79 (d, 1H, J=8 Hz), 10.74 (s, 1H).

To a solution of the acid (0.47 g, 2.1 mmol) in dry methanol (40 ml) wasadded conc. Sulfuric acid (0.47 g) and the solution heated to reflux for96 h. Upon cooling the methanol was removed and the residue taken up inwater (50 ml), and extracted with dichloromethane (3×30 ml). Thecombined extracts were dried (Na₂SO₄), and the solvent removed. Theresidue was eluted through a silica column using 5% ethyl acetate inpetroleum spirit to yield a colorless oil (0.23 g, 46%). ¹H-NMR (CDCl₃,300 MHz): δ 3.94 (s, 3H), 4.03 (s, 2H), 6.81 (t, 1H, J=8 Hz), 7.20-7.32(m, 6H), 7.73 (dd 1H, J=8 Hz, 1.5 Hz), 11.10 (s, 1H).

(4-Morpholinyl)-3-[2′-hydroxy-3′-(phenylmethyl)phenyl]-3-oxopropanamide

A cooled solution of diisopropylamine (0.62 ml, 4.4 mmol) intetrahydrofuran (10 ml) was treated with n-butyl lithium in hexane (1.6M, 2.73 ml, 4.4 mmol) and the solution stirred for 10 minutes at 0° C.4-Acetylmorpholine (0.25 ml, 2.2 mmol) was added and stirring wascontinued at 0° C. for a further 30 minutes. Methyl3-(phenylmethyl)salicylate (0.33 g, 1.4 mmol) in tetrahydrofuran wasadded dropwise and the mixture allowed to come to RT and stirring wascontinued overnight. The solution was neutralised with 1N HCl, and themixture extracted with dichloromethane (3×30 ml). The combined extractswere dried (Na₂SO₄) and the solvent removed. The residue was elutedthrough a silica column with 0-10% methanol in dichloromethane to yielda pale yellow oil (0.55 g), which contained residual 4-acetylmorpholine.The product was not further purified but reacted as follows.

2-morpholinyl-8-(phenyl)methyl)-4H-1-benzopyran-4-one (TGX-90)

To a stirred solution of the partially purified(4-morpholinyl)-3-[2′-hydroxy-3′-(phenylmethyl)phenyl]-3-oxopropanamide(0.55 g) in dichloromethane under nitrogen was added dropwisetrifluoromethanesulfonic anhydride and the solution was stirred at RTovernight. The solvent was removed, and the residue taken up in methanol(10 ml) and stirring continued for a further 4 h. The methanol wasremoved and the residue treated with half saturated sodium bicarbonatesolution (30 ml), and extracted with dichloromethane (3×20 ml). Thecombined extracts were washed (sat. NaCl), dried (Na₂SO₄) and thesolvent removed to yield an orange solid, which was recrystallized fromethyl acetate to yield pale pink, fine needles (0.12 g, 27% from 3).¹H-NMR (CDCl₃, 300 MHz): δ 3.32 (t, 3H), 3.69 (t, 3H), 4.19 (s, 2H),5.47 (s, 1H), 7.13 (d, 1H, J=8 Hz), 7.20-7.40 (m, 6H), 8.08 (dd 1H, J=8Hz, 1.8 Hz).

EXAMPLE A-2 2-(4-morpholinyl)-8-phenoxy-4H-1-benzopyran-4-one (TGX-134)Methyl 2,3-dihydroxybenzoate

A mixture of 2,3-dihydroxybenzoic acid (3.8 g, 24.6 mmol) in methanol(300 ml) was treated dropwise with conc. Sulfuric acid (4.2 g) and theresultant solution was heated at reflux temperature overnight. Uponcooling the solvent was evaporated and the residue poured intoice-water. The mixture was extracted with dichloromethane (3×50 ml) andthe combined organic fractions dried (Na₂SO₄) and concentrated to yielda pale tan solid (4.05 g).

¹H-NMR (CDCl₃, 400 MHz): δ 3.92 (s, 3H), 6.76 (t, 1H, J=7.6 Hz), 7.08(d, 1H, J=7.2 Hz), 7.33 (d, 1H, J=7.6 Hz), 10.88 (s, 1H).

Methyl 3-phenoxy-2-hydroxybenzoate

To a mixture of methyl 2,3-dihydroxybenzoate (1.50 g, 8.9 mmol),phenylboronic acid (1.08 g, 8.9 mmol) and copper acetate (1.62 g, 8.9mmol) suspended in dichloromethane (100 ml) was added triethylamine(6.15 ml, 44.5 mmol) and the mixture was stirred at room temperature for96 h. The solvent was removed and the product chromatographed through asilica column using a gradient of 0-10% methanol in dichloromethane. Theproduct was obtained as a pale yellow oil (0.25 g).

¹H-NMR (CDCl₃, 300 MHz): δ 3.97 (s, 3H), 6.86 (t, 1H, J=8 Hz), 6.9-7.4(m, 6H), 7.67 (dd, 1H, J=8 Hz, 2 Hz), 10.94 (s, 1H).

2-(4-morpholinyl)-8-phenoxy-4H-1-benzopyran-4-one (TGX-134)

Condensation of the lithium enolate of N-acetyl morpholine (0.21 g, 1.6mmol) with methyl 3-phenoxy-2-hydroxybenzoate (0.25 g, 1.0 mmol)followed by cyclodehydration with trifluoromethanesulfonic anhydride(0.60 ml, 3.6 mmol) as described above yielded TGX-134 as an off-whitesolid (0.090 g).

¹H-NMR (CDCl₃, 300 MHz): 3.22 (t, 4H, 6 Hz), 3.63 (t, 4H, 6 Hz), 5.46(s, 1H), 6.97 (d, 2H, J=9 Hz), 7.09 (t, 1H, J=8 Hz), 7.2-7.4 (m, 4H),7.94 (dd, 1H, J=6 Hz, 4 Hz)

EXAMPLE A-32-(4-morpholinyl)-8-trifluoromethanesulflonyloxy-4H-1-benzopyran-4-oneMethyl 2-hydroxy-3-trifluoromethanesulfonyloxy-benzoate

To methyl 2,3-dihydroxybenzoate (2.1 g, 12.5 mmol) dissolved indichloromethane (50 mL) was added pyridine (2.0 ml, 25 nmol) anddimethylaminopyridine (150 mg, 1.25 mmol). The mixture was cooled to 0°C. and trifluoromethane sulfonic anhydride was added dropwise bysyringe. The ice bath was removed and stirred at room temperature for 60h. The organic layer was washed twice with 1 M HCl (20 ml), dried(Na₂SO₄) and concentrated to dryness in vacuo. The solid wasrecrystallized from ethyl acetate to yield colourless crystals (2.5 g).

¹H-NMR (CDCl₃, 300 MHz): δ 3.99 (s, 3H), 6.93 (t, 1H, J=8.1 Hz), 7.43(d, 1H, J=8.4 Hz), 7.86 (d, 1H, J=8.1 Hz), 11.2 (s, 1H).

2-(4-morpholinyl)-8-trifluoromethanesulfonyloxy-4H-1-benzopyran-4-oneCondensation of the lithium enolate of N-acetyl morpoline (2.2 ml) withmethyl 2-hydroxy-3-trifluoromethanesulfonyloxy-benzoate (3.56 g, 11.9mmol) yielded the salicylacatamide (3.6 g). Cyclodehydration of theproduct with trifluormethanesulfonic anhydride (5.5 ml) as describedabove yielded the product as a colourless solid (1.21 g).

¹H-NMR (CDCl₃, 400 MHz): δ 3.57 (bs, 4H), 3.84 (bs, 4H), 5.52 (s, 1H),7.38 (t, 1H, J=6.8 Hz), 7.48 (d, 1H, J=8.0 Hz), 8.15 (d, 1H, J=8.0 Hz).

Method B

EXAMPLE 1-B2-(4-morpholinyl)-8-(4-fluoro-2-methylphenyl)oxy-4H-1-benzopyran-4-one(TGX-184)

2-(4-morpholinyl)-8-hydroxy-4H-1-benzopyran-4-one

To a solution of the trifluoromethanesulfonate ester (0.53 g, 1.4 mmol)in THF (25 ml) was added sodium t-butoxide (0.203 g, 2.1 mmol) and themixture stirred at room temperature overnight. The solvent was removedand the residue was directly chromatographed through a column of silica,eluting with 0-10% methanol in dichloromethane to yield a white solid(0.19 g).

¹H-NMR (d₆-DMSO, 400 MHz): δ 3.49 (s, 4H), 3.69 (s, 4H), 5.45 (s, 1H),7.10 (d, 2H, J=6.8 Hz), 7.31 (s, 1H), 10.14 (s, 1H).

2-(4-morpholinyl)-8-(4-fluoro-2-methylphenyl)oxy-4H-1-benzopyran-4-one(TGX-184)

To a mixture of 2-(4-morpholinyl)-8-hydroxy-4H-1-benzopyran-4-one (77mg, 0.31 mmol), 4-fluoro-2-methylphenylboronic acid (48 mg, 0.31 mmol)and copper acetate (57 mg, 0.31 mmol) suspended in dichloromethane (3.1ml) was added triethylamine (216 uL, 1.56 mmol) and the mixture wasstirred at room temperature for 24 h. The solvent was removed and theproduct chromatographed through a silica column with 0-10% methanol indichloromethane to yield an off white solid (37 mg).

¹H-NMR (d₆-DMSO 300 MHz): δ 2.27 (s, 3H), 3.38 (t, 4H, 5 Hz), 3.74 (t,4H, 5 Hz), 5.51 (s, 1H), 6.7-6.9 (m, 2H), 7.01 (m, 2H), 7.22 (t, 1H, J=9Hz), 8.55 (dd, 1H, J=9 Hz, 2 Hz).

In a similar manner were also synthesized:

-   8-phenoxy-2-morpholinyl-4H-1-benzopyran-4-one (TGX-134);-   8-(2-methylphenyl)oxy-2-morpholinyl-4H-1-benzopyran-4-one (TGX-182);    and-   8-(4-fluoro-3-methylphenyl)oxy-2-morpholinyl-4H-1-benzopyran-4-one    (TGX-173).    Method C

EXAMPLE C-1 2-morpholinyl-8-(4-fluorophenyl)-4H-1-benzopyran-4-one(TGX-165)

To a solution of the triflate (0.20 g, 0.52 mmol), potassium carbonate(0.182 g, 1.38 mmol) in acetonitrile (10 ml) bubbling under nitrogen,was added 4-fluorophenyl boronic acid (0.089 g, 0.63 mmol) followed bypalladium acetate (0.012 g, 0.05 mmol) and the solution was heated undernitrogen for 24 h. Upon cooling the mixture was filtered and the filtercake washed with acetonitrile (10 ml). The filtrate and washings werecombined and the solvent removed to yield a yellow solid which waseluted through a silica column using ethyl acetate yielding a colourlesssolid (0.057 g).

¹H-NMR (CDCl₃, 300 MHz): δ 3.33 (t, 4H, J=5.3 Hz), 3.74 (t, 4H, J=5.3Hz), 5.52 (s, 1H), 7.16 (t, 1H, J=10 Hz), 7.40 (t, 2H, J=8.7 Hz),7.45-7.55 (m, 3H), 8.18 (dd, 1H, J=9.0 Hz, 2 Hz).

In this manner but utilizing the appropriate arylboronic acid andtrifluoromethanesulfonate ester were prepared:

-   2-morpholinyl-8-(2-methylphenyl)-4H-1-benzopyran-4-one (TGX-145);-   2-morpholinyl-8-(2-trifluoromethylphenyl)-4H-1-benzopyran-4-one    (TGX-135);-   2-morpholinyl-8-(2-chlorophenyl)-4H-1-benzopyran-4-one (TGX-146);    and-   2-morpholinyl-8-(4-phenoxyphenyl)-4H-1-benzopyran-4-one (TGX-166).

EXAMPLE 5 In vitro PI 3-Kinase Assay

The effect of TGX-25, TGX-33, TGX-37, TGX-40, TGX-41, TGX-57, TGX-84,TGX-90, TGX-93, TGX-98, TGX-99, TGX-101, TGX-106, TGX-107, TGX-108,TGX-109, TGX-111, TGX-112, TGX-113, TGX-115, TGX-120, TGX-121, TGX-123,TGX-124, TGX-126, TGX-127, TGX-130 or TGX-131on PI 3-kinase activity wasdetermined using an in vitro PI 3-kinase assay. This assay was performedusing PI 3-kinase immunoprecipitated from human platelets as the enzymeand PI as the substrate. The PI 3-kinase activity was quantitated bymeasuring the enzymatic incorporation of [³²P] into PI, formingPI([³²P]-3)P, as previously described (Susa et al., 1992, The Journal ofBiological Chemistry 267(32):22951-22956.

Washed human platelets were lysed in Triton X-100 lysis buffer (10 mMTris, pH 7.4, 1% Triton X-100, 2 mM EDTA, 1 mM PMSF) for 30 minutes. TheTriton X-100 insoluble fraction was removed by centrifugation of thecell lysates at 15,000 g for 10 minutes. PI 3-kinase wasimmunoprecipitated by mixing 500 μg of the cell lysate with 1 μg of arabbit anti-rat antibody against the p85/110 form of PI 3-kinase and 30μl of 50% Protein A-sepharose beads for 2 hours at 4° C. The ProteinA-sepharose-bound PI 3-kinase was isolated by pelleting the beads at15,000 g for 5 seconds, and washing three times with ice-cold TritonX-100 lysis buffer followed by four washes with PI 3-kinase assay buffer(20 mM HEPES, pH 7.4, 1 mM EGTA, 5 mM MgCl₂).

PI stored in CHCl₃ was dried under N₂, resuspended in the lipid buffer(50 mM HEPES, pH 7.2, 1 mM EDTA) at a final concentration of 330 μg/ml,and sonicated for 6 minutes on ice. PI([³²P]-3)P was generated by mixingthe immunoprecipitated PI 3-kinase for 20 minutes with 40 μl of the PI,10 μl of ATP (1 mM) and ³²P-r-ATP (0.5 μCi, 1 μCi/nmol), 10 μl of 10×kinase buffer, in a final assay volume of 100 μl adjusted with H₂O.TGX-25, TGX-33, TGX-37, TGX-40, TGX-41, TGX-57, TGX-84, TGX-90, TGX-93,TGX-98, TGX-99, TGX-101, TGX-106, TGX-107, TGX-108, TGX-109, TGX-111,TGX-112, TGX-113, TGX-115, TGX-120, TGX-121, TGX-123, TGX-124, TGX-126,TGX-127, TGX-130 or TGX-131 was preincubated with the PI 3-kinase for 5minutes prior to the addition of ATP. The assay was terminated with 100μl of 1 N HCl, and the PI([³²P]-3)P product extracted with 200 μlchloroform:methanol (1:1) and 500 μl 2 M KCl. The PI([³²P]-3)P in thechloroform phase was resolved by thin layer chromatography using asolvent system containing CHCl₃:MeOH:HAC:H₂O (43:38:5:7) (v:v:v:v), andvisualized by autoradiography. The PI([³²P]-3)P spots were then scrapedoff from the TLC plates, deacylated with 1 mlmethylamine:butanol:methanol (42:9:47) (v:v:v) for 4 hours at 53° C.,and quantitated using a liquid scintillation counter (LKB 1209RackBETA).

The inhibitory concentration (μM) for each of the tested compounds islisted in Table IV below.

TABLE IV Compound IC₅₀ (μM) TGX-25 ~11.1 TGX-37 ~10.5 TGX-40 ~1.5 TGX-41~9.8 TGX-57 2 TGX-84 0.1 TGX-90 0.1 TGX-93 1 TGX-98 1 TGX-99 1 TGX-1010.1 TGX-102 0.1 TGX-106 2 TGX-107 1.0 TGX-108 1 TGX-109 1 TGX-111 0.05TGX-112 0.5 TGX-113 0.5 TGX-115 0.05 TGX-118 10.0 TGX-120 1.0 TGX-1210.05 TGX-123 0.2 TGX-124 1.0 TGX-125 25 TGX-126 0.05 TGX-127 0.05TGX-130 0.2 TGX-131 0.5 TGX-132 1.0 TGX-133 5.0 TGX-134 0.1 TGX-135 0.2TGX-137 0.05 TGX-138 1.0 TGX-139 1.0 TGX-140 10.0 TGX-141 1.0 TGX-1422.0 TGX-143 0.15 TGX-144 2.0 TGX-145 2.0 TGX-146 0.5 TGX-147 5.0 TGX-14810.0 TGX-149 0.5 TGX-151 0.5 TGX-152 0.5 TGX-153 20.0 TGX-154 10.0TGX-155 0.02 TGX-156 5.0 TGX-157 5.0 TGX-158 5.0 TGX-159 10.0 TGX-1602.0 TGX-161 0.5 TGX-162 2.0 TGX-163 1.0 TGX-165 1.0 TGX-167 0.05 TGX-1680.75 TGX-169 7.5 TGX-170 0.2 TGX-173 0.1 TGX-174 0.1 TGX-176 0.5 TGX-17910.0 TGX-180 1.0 TGX-186 0.01

EXAMPLE 6 Flow-Based Reconstitution Assay

The effect of TGX-40 on platelet adhesion was examined using aflow-based adhesion assay. Washed platelets were pretreated with 10, 25,or 50 μM TGX-40, or control buffer (0.1% DMSO) for 30 minutes at 37° C.prior to reconstitution with red blood cells to a hematocrit of 50%. Theplatelets and reconstituted red blood cells were perfused throughvWf-coated glass microslides for 1 minute at a shear rate of 1800 s⁻¹.Non-adherent cells were removed by washing for 10 minutes at 1800 s⁻¹and the number of adherent platelet were quantitated and expressed asthe mean±SEM. As illustrated graphically in FIG. 1, TGX-40 inhibited theability of platelets to adhere in a dose-dependent manner, showing adecrease of 51, 67 and 86% in platelet adhesion when platelets werepretreated with 10, 25, and 50 μM TGX-40.

EXAMPLE 7 Whole-Blood How Assay

The inhibitory effect of TGX-40 on platelet thrombus formation wasexamined using a whole-blood flow assay, since thrombi formed by washedplatelets are small and poorly reproducible. Anticoagulated whole bloodwas incubated with 50, 100, or 200 μM TGX-40, or control buffer (0.1%DMSO) for 30 minutes with gentle rocking prior to perfusion throughvWf-coated glass microslides for 2 minutes at a shear rate of 1800 s⁻¹.Non-adherent platelets were removed by washing for 10 minutes at 1800s⁻¹, and adherent erythrocytes were lysed with 1% ammonium oxalate. Thelevel of thrombus formation was quantitated indirectly by measuringplatelet LDH (U/L) levels in the whole cell lysates byspectrophotometry. Following a 2-minute perfusion of whole blood,platelet-rich thrombi were observed over the surface of the microslide.As seen in the photographs of FIG. 2, pretreatment with TGX-40 inhibitedthe ability of platelet thrombi to form on the vWf matrix in adose-dependent manner. As illustrated graphically in FIG. 2,pretreatment of whole blood with 50, 100, and 200 μM TGX-40 led to adecrease of 25, 53, and 80% in thrombus formation relative to control.

EXAMPLE 8 Animal Model of Internal CarotidArtery Occlusion

The inhibitory effect of TGX-40 was examined in the well establishedanimal model of arterial thrombosis of Folts et al., 1982, Circulation65:248-255. This model is used to investigate the effects ofantithrombotic drugs on clotting time in vivo in response to a crushinjury followed by arterial stenosis.

The carotid artery of an anesthetized rat is dissected out, and anelectromagnetic flow probe is placed around the artery to measure bloodflow. Proximal to the flow probe, the artery is clamped with surgicalforceps covered with silicone tubing to cause intimal and medial damageto the vessel wall. A ligature, or plastic cylinder of appropriateinside diameter is laced around the artery to produce a 70% reduction inarterial diameter.

Platelets aggregate in the area of the stenosed and damaged arterialvessel, gradually forming an occlusive platelet thrombus, seen as adecrease in blood flow. As the thrombus forms, blood pressure increases,causing the thrombus to fragment and embolize distal to the stenosedsite. If the thrombus does not embolize spontaneously, the stenosedregion is shaken gently to dislodge the thrombus. This causes a suddenrestoration of blood flow. Platelets again aggregate in the area of thestenosed and damaged arterial vessel, repeating thethrombus-embolization pattern. This acute platelet-mediated thrombusformation, followed by embolization, causes Cyclic Flow Reductions (CFR)in blood flow. Once a rat produces regular CFRs, an anti-thromboticcompound or vehicle control is administered via the jugular vein.

TGX-40 was administered at doses of 1.6 mg/kg and 3.2 mg/kg via thejugular vein and the stabilization of blood flow was recorded. Asillustrated graphically in FIG. 3, TGX-40, at 1.6 mg/kg and 3.2 mg/kg,returned 90% of the treated animals to baseline within 10 minutes,indicating that the compound has utility in the treatment of coronaryartery occlusion.

EXAMPLE 9 Effect of TGX-84 on Platelet Thrombus Formation Under Now

Citrated whole blood was pretreated with 50, 100 or 200 μM TGX-84, orcontrol buffer (0.1% DMSO) for 10 minutes at 37° C. Blood was perfusedthrough von Willebrand factor-(vWf) coated microcapillary tubes for 2minutes at 600 s⁻¹. Non-adherent cells were removed by perfusion ofbuffer for 2 minutes at 600 s⁻¹ and any adherent erythrocytes lysedthrough treatment with 1% ammonium oxalate. Adherent platelets were thenlysed through addition of 1% Triton X-100 and lactate dehydrogenase(LDH) levels (U/L) analysed by spectrophotometry. The results aregraphically shown in FIG. 4. As illustrated in FIG. 4, pretreatment ofwhole blood with 50, 100, 200 μM TGX-84 led to a decrease in thrombosisformation relative to control.

EXAMPLE 10 In vitro Enzyme Assays PI3K and PI4K

In vitro enzyme assays were conducted as a primary screen to determinedrug candidate isoform affinity and specificity. The affinity of twoleading compounds of the quinolone series (TGX84 and TGX155) for aclosely related enzyme family, P14K, was also determined to maximisecompound specificity and therefore minimise potential adversebiochemical events.

The α and β isoforms of the PI3K were immunoprecipitated from a plateletlysate using an antibody that recognised the p85 regulatory subunitcommon to both forms of the enzyme. The γ isoform was produced as arecombinant protein in the Thrombogenix laboratories. PI4K was isolatedfrom platelets in a similar manner using a PI4K specific antibody.Standard phosphorylation assays using phosphatidylinositol and ³²P wereused to measure the enzyme activity in the immunoprecipates in thepresence or absence of an inhibitor. Enzyme activity was determined overa range of inhibitor concentrations to establish an IC₅₀ value.

The IC₅₀ for LY294002 against the α/β isoforms of PI3K was in agreementwith previously reported values (1-1.5 μM).

TABLE V Affinity of LY294002 and Thrombogenix compounds for PI3K α/βisoforms Compound Chemical class PI3K α/β IC₅₀ (μM) PI3K γ (μM) LY294002— 1-1.5 2 TGX-155 QU 0.02 5 TGX-127 QU 0.05 5-10 TGX-115 QU 0.05 5TGX-167 PP 0.05 5-10 TGX-137 PP 0.05 5 TGX-126 PP 0.05 >10 TGX-183 PP0.05 TGX-184 BP 0.05 TGX-121 QU 0.05 5 TGX-111 QU 0.05 >10 TGX-84 QU 0.15 TGX-101 PP 0.1 2 TGX-174 PP 0.1 5 TGX-134 BP 0.1 0.2 TGX-102 BP 0.1 2TGX-90 BP 0.1 3 TGX-143 QU 0.15 2 TGX-173 BP 0.15 QU—quinolone series;PP—pyridopyrimidine series; BP—benzopyranone series.

In contrast to its highly potent affinity for PI3K, TGX155 and TGX84exhibited an IC₅₀ of 100 μM against PI4K.

EXAMPLE 11 Enzyme Screening Assay

The two leading compounds of the quinolone series, TGX155 and TGX84 werescreened for activity against seven enzymes related to PI3K in functionor substrate specificity, viz: ATPase, PDE4, tyrosine kinases EGF andfyn, protein kinases A and C, and tyrosine phosphatase. The IC₅₀ valuesfor TGX155 and TGX84 inhibition of each enzyme were greater than 100□μM,confirming the target specificity of the compounds.

EXAMPLE 12 Cell Proliferation Assay

The anti-proliferative activity of the compounds of this invention fromall three chemical classes was determined using K562 (leukaemia derived)and U937 (moncytic) cell lines. The cytotoxic activity of the compoundswas monitored over four days by counting cell number and determiningcell viability using a colourimetric assay metabolic activity.

Antiproliferative Activity of TGX Compounds (20 μM, 4 day incubation)Compound % Cells remaining TGX-168 15 TGX-123 10 TGX-167 1.5 TGX-186 1.5TGX-40 75

These data demonstrate that the compounds are useful in preventing cellproliferation. Hence the compounds of this invention may be useful inthe treatment of cancer and other disorders, such as asthma, whereabnormal cell proliferation is involved.

EXAMPLE 13 Making and Administering Pharmaceutical Compositions thatContain Morpholino-Substituted Compounds

Another aspect of the present invention relates to a pharmaceuticalcomposition containing a morpholino-substituted compound of the presentinvention together with one or more pharmaceutically acceptable carriersand/or diluents. Below, the term “active ingredient” may be anymorpholino-substituted compound of the present invention, or aphysiologically acceptable salt, solvate, or functional derivativethereof.

Administration of this pharmaceutical composition is performed by anyconvenient means. Doses are administered daily, weekly, monthly, or atother suitable time intervals such as by the oral, intravenous,intraperitoneal, intramuscular, subcutaneous, intradermal, orsuppository routes, or by implanting (e.g. using slow-releasemolecules). If the active compound is administered in tablet form, thetablet contains a binder such as tragacanth, corn starch, or gelatin; adisintegrating agent, such as alginic acid; and a lubricant, such asmagnesium stearate.

The pharmaceutical compositions suitable for injectable use includesterile aqueous solutions or dispersions, and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions, or are in the form of a cream or other form suitable fortopical application. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity ismaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersion,and by the use of superfactants. Prevention of contamination bymicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal and the like. It may be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, followed by filter sterilization.Generally, dispersions are prepared by incorporating the varioussterilized active compounds into a sterile vehicle containing the basicdispersion medium and one or more of the above-described ingredients. Inthe case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze drying which yield a powder of the active compound plus anyadditional desired ingredients from previously sterile-filteredsolutions thereof.

The pharmaceutical compositions are orally administered, for example,with an inert diluent or with an assimilable edible carrier, areenclosed in hard or soft shell gelatin capsule, are compressed intotablets, or are incorporated directly with food. For oraladministration, the active compounds are incorporated with excipients,and are used in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Suchcompositions and preparations contain at least 1% by weight of activecompound. The percentage of the compositions and preparations may bevaried and may be between about 5 to about 80% of the weight of theunit. The amount of active compound in such therapeutically usefulcompositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain abinder such as gum, acacia, corn starch, or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch, alginic acid and the like; a lubricant such as magnesiumstearate; and a sweetening agent such as sucrose, lactose or saccharinmay be added or a flavoring agent such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.A syrup or elixir may contain the active compound, sucrose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompound may be incorporated into sustained-release preparations andformulations.

Some of the preferred pharmaceutical formulations of the presentinvention are described below.

Tablet Formulation for Oral Administration:

The ingredients of a tablet formulation for oral administration arelisted in Table VI below. Tablets A, B, and C are prepared by wetgranulation, with the povidone, of the first six ingredients listed inTable VI, followed by the addition of the magnesium stearate andsubsequent compression.

TABLE VI Milligrams per Tablet Tablet A Tablet B Tablet C Activeingredient 25 25 25 Avicel 13 — 7 Lactose 78 47 — Starch (maize) — 9 —Starch (pregelatinised, NF15) — — 32 Sodium starch glycollate 5 — —Povidone 3 3 — Magnesium stearate 1 1 1 Total 125 85 85Tablet Formulation for Sublingual Administration:

The ingredients of two tablet formulations for sublingual administrationare listed in Table 4 below. Tablets A and B are prepared by wetgranulation, with the povidone, of the first six ingredients listed inTable 4, followed by the addition of the magnesium stearate andsubsequent compression.

TABLE 4 Milligrams per Tablet Tablet A Tablet B Active ingredient 25 25Avicel 10 — Lactose — 36 Mannitol 51 57 Sucrose — 3 Acacia — 3 Povidone 3 — Magnesium stearate  1 1 Total 90 125Tablet Formulation for Buccal Administration:

A tablet for buccal administration is prepared by admixing theingredients listed in Table 5 below, followed by direct compression ofthe admixed ingredients.

TABLE 5 Milligrams per Tablet Active ingredient 25 Hydroxypropylmethylcellulose 25 (HPMC) — Polycarbophil 39 Magnesium stearate  1 Total 90Powder-Filled Capsule Formulation:

The ingredients of two powder-filled capsule formulations are listed inTable 6 below. Capsules A and B are prepared by admixing theingredients, and filing two-part hard gelatin capsules with theresulting mixture.

TABLE 6 Milligrams per Tablet Capsule A Capsule B Active ingredient 25 —Avicel 45 — Lactose 153 — Starch (1500 NF) — 117 Sodium starchglycollate — 6 Magnesium stearate 2 2 Total 225 150Liquid-Filled Capsule Formulation:

The ingredients of two liquid-filled capsule formulations are listed inTable 7 below. Capsule A is prepared by melting the Macrogol 4000 BP,dispersing the active ingredient in the melt, and filling two-part hardgelatin capsules therewith. Capsule B may be prepared by dispersing theactive ingredient in the lecithin and arachis oil, and filling soft,elastic gelatin capsules with the resulting dispersion.

TABLE 7 Milligrams per Tablet Capsule A Capsule B Active ingredient 2525 Macrogol 4000 USP 200 — Lecithin — 100 Arachis oil — 100 Total 225225Controlled-Release Capsule Formulation:

A capsule formulation for controlled release is prepared by mixing andextruding the first four ingredients listed in Table 8 below, andspheronizing and drying the extrudate. The dried pellets are coated withthe ethyl cellulose as a release-controlling membrane, and the resultingpellets are filled into two-part hard gelatin capsules.

TABLE 8 Milligrams per Capsule Active ingredient 25 Avicel 123 Lactose62 Triethyl citrate 3 Ethyl cellulose 12 Total 225Intravenous Formulation:

The intravenous formulation containing the ingredients listed in Table 9below is prepared by taking up the active ingredient in the citratebuffer, and the pH of the solution is then adjusted to pH 7 withhydrochloric acid. The resulting solution is made up to volume, and issubsequently filtered through a micropore filter into sterile glassvials which are sealed and oversealed after filling.

TABLE 9 % by weight Active ingredient 2 Hydrochloric acid (citratebuffer) q.s. to pH 7 Water for injections to 100%Intranasal Formulation:

An intranasal formulation containing the ingredients listed in Table 10below is prepared by taking up the active ingredient in a mixture of thehydroxybenzoates, and the pH of the solution is then adjusted to pH 7with hydrochloric acid in citrate buffer. The resulting solution is madeup to volume, and is subsequently filtered through a micropore filterinto sterile glass vials which are sealed and oversealed after filling.

TABLE 10 % by weight Active ingredient 0.5 Hydrochloric acid in citratebuffer q.s. to pH 7 Methyl hydroxybenzoate 0.2 Propyl hydroxybenzoate0.2 Water for injections to 100%Intramuscular-Injection Formulation:

A formulation for intramuscular injection containing the ingredientslisted in Table 11 below is prepared by dissolving the active ingredientin the glycofurol. The benzyl alcohol is then added and dissolved, andwater is added to bring the final volume to 3 ml. The mixture is thenfiltered through a micropore filter into sterile glass vials which aresealed and oversealed after filling.

TABLE 11 Active ingredient 0.05 g Benzyl alcohol 0.1 g Glycofuro 7511.45 g Water for injections q.s. to 3.00 mlSyrup Formulation:

A syrup formulation containing the ingredients listed in Table 12 belowis prepared by dissolving the sodium benzoate in a portion of purifiedwater, and the sorbitol solution is then added. Subsequently, the activeingredient is added and dissolved. The resulting solution is then mixedwith the glycerol and made up to the required volume with purifiedwater.

TABLE 12 Active Ingredient 0.05 g Sorbitol solution 1.5 g Glycerol 1.0 gSodium benzoate 0.005 g Flavor 0.0125 mlSuppository Formulation:

A suppository formulation containing the ingredients listed in Table 13below is prepared by melting one-fifth of the Witepsol in asteam-jacketed pan at a maximum temperature of 45° C. The activeingredient is then sifted through a 200 μm sieve and mixed with themolten base using a Silverson mixer fitted with a cutting head until asmooth dispersion is achieved. Maintaining the mixture at 45° C., theremaining Witepsol H15 is added to the suspension which is stirred toensure a homogenous mix. The entire suspension is then passed through a250 μm stainless steel screen and, with continuous stirring, allowed tocool to 40° C. At a temperature of between 38 and 40° C., 2.0 g aliquotsof the mixture are filled into suitable plastic molds. The resultingsuppositories are allowed to cool to room temperature.

TABLE 13 Milligrams per Suppository Active ingredient (63 μm)¹ 50 Hardfat, USP (Witepsol H15 - dynamit 1950 NoBel) Total 2000 ¹The activeingredient is used as a powder wherein at least 90% of the particles areof 63 μm diameter or less.Aerosol Formulation:

An aerosol formulation containing the ingredients listed in Table 14below is prepared by mixing the active compound with ethanol, and wateris added for injection. The solution is subsequently added to a portionof the Propellant 22, cooled to −30° C., and transferred to a fillingdevice. The required amount is then fed to a stainless steel containerand diluted with the remainder of the propellant. The valve units arethen fitted to the container.

TABLE 14 % by weight Active ingredient 0.25 Ethanol 10 Water forinjections 19.75 Propellant 22 (chlorodifluoromethane) 70 Total 100Pessary Formulation:

A pessary formulation is prepared by directly mixing the ingredientslisted in the Table 15 below. Pessaries are prepared by compressing theresulting mixture.

TABLE 15 Milligrams per Pessary Active ingredient (63 μm)¹ 50 Anhydrousdextrose 470 Potato starch 473 Magnesium stearate 473 Water forinjections 1000 ¹The active ingredient is used as a powder wherein atleast 90% of the particles are of 63 μm diameter or less.

1. A compound having the following formula:

R is H, OH, F, Cl, Br, I, C₁-C₆ alkyl, aryl or (CH₂)_(n)-aryl; one of R¹and R² is H or C₁-C₆ alkyl, and the other is O-aryl, wherein aryl isoptionally substituted with F, Cl, Br, I, CN, CO₂H, CO₂R³, NO₂, CF₃,substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted aryl, OCF₃, OR³, OSO₂-aryl,substituted or unsubstituted amine, NHCOR³, NHSO₂R³, CONHR³, or SO₂NHR³;and R³ is H, or substituted or unsubstituted C₁-C₆alkyl, substituted orunsubstituted aryl.
 2. The compound of claim 1, wherein R¹ is selectedfrom a group consisting of —CH₃,